The acetylcholine (ACh) receptor is one of the best characterized of the membrane proteins involved in excitability phenomena. The functional characterization of this receptor has been helped by the discovery of very specific toxins with novel actions and high receptor- binding affinities. We have recently demonstrated cholinergic activity in extracts obtained from ciguatoxic barracuda and in extracts, as well as in chromatographically isolated fractions, obtained from Ostreopsis lenticularis - the benthic dinoflagellate associated with ciguatera in the Caribbean. Methanolic extracts inhibited synaptic potentials and the contractions elicited by nerve-released or bath-applied acetylcholine in frog sartorious muscles. This effect appeared to be caused by a polar fraction in the dinoflagellate material that was capable of reducing the ionic conductance of nicotinic ACh receptor channels in cultured chick embryo myocytes. Other studies showed that extracts and a second chromatographic fraction displayed muscarinic activity, competing with the binding of tritiated muscarinic ligands to rat brain membranes and exhibiting an agonist-like action in frog gastric muscularis muscles. This application aims to discover the mechanism of the cholinergic effects of these dinoflagellate toxins using a variety of electrophysiological, pharmacological and biochemical techniques. Experiments are proposed to attain the following specific aims: a) the chromatographic isolation of the fractions from toxic extracts of the Caribbean dinoglagellate Ostreopsis lenticularis using reverse phase HPLC; b) the determination of the chemical nature and structure of cholinoactive components; and c) identification of the sites and mechanisms of action of the isolated cholinergic - receptor active compounds. Research strategies to be used in this latter phase are: i) binding experiments using neuronal membranes and membranes from Torpedo electric organ to examine displacement of and competition with radiolabeled ligands of the different receptor subtypes; ii) intracellular recording of synaptic potentials in frog motor end plates and measurement of changes in their magnitude and temporal characteristics caused by the active fractions; and iii) recording of membrane ionic currents, using both the single channel and the whole cell versions of the patch clamp technique in cultured chick embryo myocytes and neurons from sympathetic, ciliary and dorsal root ganglia. Once identified and characterized these new dinoflagellate toxins should be useful molecular probes for the study of cholinergic receptors in the central and peripheral nervous systems. Minority students participating in these studies will learn modern neurochemical and electrophysiological methods as well as tissue culture techniques.